Etiologic Risk Group: Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Background/Disease information: The disease involved with this gene/protein (Fab G-ketoacyl-(acyl-carrier-protein) reductase) is epidemic typhus, which is caused by Rickettsia prowazekki bacteria. This disease spreads to humans through lice or ticks and can be found on the flying squirrel. Although uncommon in the US, several cases of typhus have been reported in Texas, in particular in Austin. Since the US is hygienically advanced, it is less prone to exposure and spread of typhus; but areas of the world with lower hygiene and cold temperature, like certain third world countries, are much more likely to have a large epidemic due to typhus. After contraction, the disease may be dormant for years before it re-activates. Death may occur, if left untreated, in ten to sixty percent of patients. Symptoms include the following:
severe headache
high fever
rash that begins on the back or chest/spreads
confusion
stupor and seeming out of touch with reality
low blood pressure
eye sensitivity to bright lights
severe muscle pain
Common treatments include antibiotics like tetracycline and the disease can be prevented by bathing, avoiding infested areas, and sterilizing infected clothing.
Enzyme Assay Information: (3R)-3-hrdroxyacyl-[acyl-carrier protein] plus NADP+; spectrophotometric
Substrate Info-100113
NADPH Preparation Instructions β-NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution.
Storage/Stability It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 °C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are β-NADP and monophosphoadenosine 5′- diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, it has been reported that a 0.5 mM solution in 0.02 M NaOH (pH 12.3) showed no loss of purity in a week at 4 °C or −85 °C, but a 13% loss at –20 °C.3
Structure Available (PDB or Homology model) PDB: 3F91
NCBI Gene # or RefSeq#: 883564
Protein ID (NP or XP#) or Wolbachia#: NP_221114.1
Organism (including strain): Rickettsia prowazekii
Etiologic Risk Group: Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Background/Disease information:
The disease involved with this gene/protein (Fab G-ketoacyl-(acyl-carrier-protein) reductase) is epidemic typhus, which is caused by Rickettsia prowazekki bacteria. This disease spreads to humans through lice or ticks and can be found on the flying squirrel. Although uncommon in the US, several cases of typhus have been reported in Texas, in particular in Austin. Since the US is hygienically advanced, it is less prone to exposure and spread of typhus; but areas of the world with lower hygiene and cold temperature, like certain third world countries, are much more likely to have a large epidemic due to typhus. After contraction, the disease may be dormant for years before it re-activates. Death may occur, if left untreated, in ten to sixty percent of patients. Symptoms include the following:
- severe headache
- high fever
- rash that begins on the back or chest/spreads
- confusion
- stupor and seeming out of touch with reality
- low blood pressure
- eye sensitivity to bright lights
- severe muscle pain
Common treatments include antibiotics like tetracycline and the disease can be prevented by bathing, avoiding infested areas, and sterilizing infected clothing.Link to TDR Targets page: not applicable
Link to Gene Database page
http://www.ncbi.nlm.nih.gov/gene/883564
Essentiality of this protein
super essential in biosynthesis (fatty acids)
structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 A res
Complex of proteins: tetramer
Druggable Target (list, number, or cite evidence from a paper/database showing druggable in another organism):
EC#: 1.1.1.100
Link to BRENDA EC#:
Enzyme Assay Information:
(3R)-3-hrdroxyacyl-[acyl-carrier protein] plus NADP+; spectrophotometric
Substrate Info-100113
NADPH
Preparation Instructions
β-NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution.
Storage/Stability
It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 °C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are β-NADP and monophosphoadenosine 5′- diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, it has been reported that a 0.5 mM solution in 0.02 M NaOH (pH 12.3) showed no loss of purity in a week at 4 °C or −85 °C, but a 13% loss at –20 °C.3
Structure Available (PDB or Homology model)
PDB: 3F91
Current Inhibitors
Flavanoids, EGCG in green tea
Expression Information
in E. Coli cells
Purification Method:
Nickel + FPLC
Image of protein
Amino Acid Sequence
MIDLTGKTSLITGASSGIGSAIARLLHKLGSKVIISGSNEEKLKSLGNALKDNYTIEVCNLANKEECSNLISKTSNLDILVCNAGITSDTLAIRMKDQDFDKVIDINLKANFILNREAIKKMIQKRYGRIINISSIVGIAGNPGQANYCASKAGLIGMTKSLSYEVATRGITVNAVAPGFIKSDMTDKLNEKQREAIVQKIPLGTYGIPEDVAYAVAFLASNNASYITGQTLHVNGGMLMVX
Length of protein:
242
Molecular Weight in kiloDaltons:
25870.1 kD
Molar Extinction coefficient of your protein at 280 nm wavelength:
10680 M-1 cm-1, 10430 M-1 cm-1
CDS Gene Sequence
CAAACAATATTTAAAAGAACATCCACAAATTTCCAACGAAATTGAGAAAATAATACGAGAAAAATCGTCAGCAATTACTAATATAAATCTTGATCAAACGGAGGAATAAAATGATTGATTTAACAGGTAAAACTTCCTTAATTACAGGTGCTTCAAGTGGCATAGGAAGTGCCATAGCGCGGTTACTACATAAACTCGGGAGCAAGGTAATTATTAGCGGTAGTAATGAAGAAAAATTAAAATCACTTGGCAATGCTTTAAAAGATAATTATACCATAGAAGTATGTAATCTTGCGAATAAGGAAGAATGTAGTAATTTAATATCTAAAACATCAAATTTAGATATTTTAGTATGTAATGCCGGTATTACTAGTGATACACTCGCAATTAGAATGAAAGATCAAGATTTCGACAAAGTTATTGATATTAATTTGAAAGCCAATTTTATTTTAAATCGTGAAGCAATAAAAAAAATGATACAAAAAAGGTATGGACGCATTATTAACATATCTTCAATAGTAGGAATTGCAGGTAACCCAGGACAGGCTAATTATTGCGCTTCTAAAGCAGGTTTAATAGGTATGACCAAATCGCTATCTTACGAAGTTGCAACTAGAGGGATTACGGTTAATGCAGTAGCACCAGGATTTATTAAATCAGATATGACTGATAAACTCAATGAAAAACAACGAGAAGCAATAGTACAAAAAATTCCACTAGGTACTTATGGTATACCAGAAGACGTTGCATACGCAGTCGCATTTTTAGCAAGCAATAATGCATCATATATTACTGGTCAAACTCTACATGTAAACGGTGGTATGTTAATGGTATAAAATTTTATGCTAGCAAAAAATAATTTTTTGTGCTAGAAGTTCTTTTAAAGAGAAAATGCTTATGAATATTTATTTCAAATATTCATAGCAATGAAAATAAAATCTAAT
GC% Content for gene:
30.6 %
CDS Gene Sequence (codon optimized)
ATGATTGACCTCACGGGCAAAACTTCTCTGATCACGGGTGCCAGCTCTGGTATCGGTTCTGCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTATCTCTGGTTCTAACGAAGAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGTTTGTAACCTCGCGAACAAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTGGTTTGCAATGCGGGCATCACCTCTGACACCCTGGCGATCCGTATGAAAGACCAGGACTTTGACAAAGTCATCGATATCAACCTGAAAGCGAACTTCATCCTGAACCGTGAAGCGATCAAGAAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCTATCGTTGGTATTGCGGGTAACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAATCTCTGTCTTACGAAGTTGCGACCCGCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTCATCAAATCTGACATGACTGACAAGCTGAATGAAAAACAGCGTGAGGCAATCGTTCAGAAAATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTACGCGGTTGCGTTCCTGGCGTCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGATGGTTTAA
GC% Contect for gene:
48.6 %
Primer Design results for pNIC-Bsa4 cloning:
1 ATGATTGACCTCACGGGCAAA 21
2 GCAGAACCGATACCAGAGCTGGCACCCGTGATCAGAGAAGTTTTGCCCGTGAGGTCAATC 60
3 GCTCTGGTATCGGTTCTGCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTA 60
4 GAGAGACTTCAGTTTTTCTTCGTTAGAACCAGAGATAATAACTTTAGAGCCCAGTTTGTG 60
5 TAACGAAGAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGT 60
6 ATCAGATTGCTGCATTCTTCTTTGTTCGCGAGGTTACAAACTTCGATGGTGTAGTTGTCT 60
7 AAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTGGTTTGCAAT 60
8 CTTTCATACGGATCGCCAGGGTGTCAGAGGTGATGCCCGCATTGCAAACCAGGATGTCCA 60
9 CCTGGCGATCCGTATGAAAGACCAGGACTTTGACAAAGTCATCGATATCAACCTGAAAGC 60
10 CATCTTCTTGATCGCTTCACGGTTCAGGATGAAGTTCGCTTTCAGGTTGATATCGATGAC 60
11 CGTGAAGCGATCAAGAAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCT 60
12 GTTCGCCTGACCTGGGTTACCCGCAATACCAACGATAGAGGAGATGTTGATAATACGACC 60
13 ACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAATCTC 60
14 CGTTGACGGTGATACCGCGGGTCGCAACTTCGTAAGACAGAGATTTGGTCATGCCGATCA 60
15 GCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTCATCAAATCTGACATGACTGACAAGC 60
16 GATTTTCTGAACGATTGCCTCACGCTGTTTTTCATTCAGCTTGTCAGTCATGTCAGATTT 60
17 TGAGGCAATCGTTCAGAAAATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTA 60
18 TGTAAGACGCGTTGTTAGACGCCAGGAACGCAACCGCGTAAGCCACGTCTTCTGGAATAC 60
19 CGTCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGA 60
20 TTAAACCATCAGCATACCACCGTTAACAT 29
Primer design results for tail primers
forward
5’ TACTTCCAATCCATGATTGACCTCACGGGCAA 3'
reverse
5’ GTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’